ISSN (online): 2320-4257
3(1):579-262
www.biolifejournal.com
AN INTERNATIONAL QUARTERLY JOURNAL OF BIOLOGY & LIFE SCIENCES
BIOLIFE
RESEARCH
A RTICLE
Evaluation of antibacterial activity of Aspergillus and Penicillium spp.
Seyedeh. Fatemeh. Miri.1* and Mohammad Ali Tajick Ghanbary 2; Heshmatollah Rahimian3 and
Ghorban Ali Nematzadeh4
1,2,3
Department of plant protection, Sari Agricultural Sciences and Natural Resources University, Sari,
Mazandaran , Iran.
4
Department of plant breeding,sari agricultural Sciences and Natural Resources University, Sari,
Mazandaran, Iran.
E-mail: [email protected]
ABSTRACT
Bacteria have known as one of the main important limitation factors of many agricultural products.
The purpose of the present study was to evaluate the antibacterial activities of different species of
Penicillium and Aspergillus on some important bacteria. For this intention 12 and 21 species of
Aspergillus and Penicillium, were applied, respectively. Gram-negative bacteria conducted in this
research were Pseudomonas viridiflava, Xanthomonas campestrispv. Campestris and Escherichia coli
and the only gram-positive bacterium was Rhatayibacter iranicus. Fungal and bacterial isolates were
dual cultured simultaneously on Potato Dextrose Broth (PDB) and were shaked with 120 rpm. The
samples were investigated after a week. The results of this study demonstrated that none of fungal
species could inhibit E. coli and the most of the studied species couldn’t suppress the growth of P.
viridiflava except isolates of (5033, 5037, and 5071) from P. crysogenum. On the other hand, A.
awamori, A. ferreus, A. ostiatus, A. niger, A. foetidus, P. mali, P. crustaseum and P. citrinumcould
lead to inhibition of X. campestris pv. campestris Gram positive bacterium, Rhatayibacter iranicus,
was not able to grow in the presence of all species of Aspergillus. On the contrary, species of P.
piscarium, P. corylophilum, P. griseofulvum and P.chrysogenum couldn’t prevent its growth.
Key words: Antibacterial, Penicillium, Aspergillus.
INTRODUCTION
Soil
biological activities occur by aerobic
unicellular where small parts of these operations
are conducted by anaerobic microorganisms
which often reside in deep depths and layers.
Aerobic microbes are gathered in upper layers of
soil where humus and organic matters exist.
Likewise, much value of oxygen is present in
underground which demonstrates maximum of
activity. It is much in number because of this
layer where too many orgasms are established
and placed. With the reduction of oxygen and
257| © www.globalsciencepg.org
organic matters from upper layers, activity and
value of microorganisms fail in underground
level. In biological definition of soil, we can
assess biological activity through live creatures
in soil. A part of biological activities in soil
result from fungi. (Mc Laughlin, 2001).
Microscopic fungi make its life activity apparent
through missile. Their distribution is done
through producing Spore. With the rise of
organic matters especially nitrogen in the
environment they increase in number. Value of
fungi in fertile arable lands is usually less than
bacteria
and
actinomycetes
and
its
environmental reaction is different depending on
Biolife | 2015 | Vol 3 | Issue 1
Seyedeh. Fatemeh. Miri et al
different seasons of year. Their maximum value
is usually observed in spring and fall. Most of
them are resistant to dryness and can also do
their operations in acid condition completely.
Totally they are more in value in humid soil
compared to semi-dry and dry areas. Among
microscopic fungi some live in form of parasite
and generating diseases and other ones live in
saprophytes form on remaining of organic
matters. Fungus of Aspergillus and Penicillium
are the most common ones which are found
nearly in all areas of dry soil to humid area.
(Alexopoulos, 1996). Soil bacteria are
unicellular organisms with plan origin on which
there is microscopic dimensions (1-10micron)
and they can be compared with particles of clay.
Adjacent to root of organic plants and in fresh
organic matters, they grow more and can live
much longer. Depending on value of soil they
reach to 2 billion in each gram. Usually their
value and activity depend on PH of environment.
If the best reactive environment for bacteria is
almost neutral, they can find good place for
living in calcareous soils and soils where there is
sufficient amount of cation and they are
propagated fast. In order to investigate biology
of soil it seems necessary to separate, identify
and study each of microorganisms available in
soil (Lotfi, 2010). In many biological and
biochemical sources of soil, enzyme activities of
soil are studied which can be applied as an index
for measuring biological activity of soil.
(Kossem, 1995).
Probably all the microorganism studies do have
enzyme activity which caused to such
conclusion. Separation and purifying secondary
metabolic of soil and assessing them in soil is
that hard. Hence, laboratory studies seem simple
and valid in producing secondary metabolic
applying pure culture. Among various secondary
metabolic groups, biologists are into antibiotics
as inhibitors of growth microorganism more than
others. Aspergillus and Penicillium fungi are
main antibiotic producers which are able to
produce and can influence on other
microorganisms in different areas. (McLaughlin,
2001). Natural compounds were the most
important sources of antibiotics or antibiotic
prototypes in the past (Butler, 2006). Despite
258 |© 2015 Global Science Publishing Group, USA
Copyright@2014
wide expectations of syntheses molecules with
effective antibiotic features, natural compounds
still promise valuable factors. (Newman &
Cragg, 2007). A research was done regarding
new strains for microorganisms, but their
antibiotic activities were not assessed yet which
can produce new molecules or useful samples
for developing antibiotics (Sofia & Boldi, 2006).
Antibiotics produced were separated from
microorganisms and most of different residents
such as Plant Endophytic (Gunatilaka, 2006) and
marine organisms (Gandhimathi, 2008).
Microorganisms are so significant in soil for
biochemical cycle, since they lead to biological
changes in the environment. Qualitative and
quantitative approaches of assessing fungi
present in soil are described well (Rousk &
Baath, 2007).
Too much fungi extractions and extracellular
components were found with antimicrobial
activity most of which were separated from
filamentous Penicillium fungus. Since the
discovery of Penicillium fungus are known as
antibiotic producers and other secondary
metabolic with biological activity.
Filamentous fungus Penicillium produces
antibacterial matters in soil which can lead to
removal of some bacteria in soil. In this research
we dealt with the ability of production of
antibacterial agents and prevention of grampositive and negative bacteria growth which had
been removed from Iran`s agricultural soils.
Using isolate stimulation, we dealt with
production of antibiotics by adding bacteria to
fungus culture.
MATERIALS AND METHODS
Fungi and bacteria sued in this research were
separated from soil of arable land of Iran and
using key they were identified. Likewise, 5
isolates of P chrysogenum were bought from
collection center of fungi and industrial bacteria
of Iran.
First fungi were cultured in PDA (Potato
Dextrose Agar) then spore suspension was
prepared and the concentration of spores was
Biolife | 2015 | Vol 3 | Issue 1
Seyedeh. Fatemeh. Miri et al
Copyright@2014
measured. For spore suspension preparation,
fungi were cultured on PDA media and after
fungal growth and spore production, 5 ml of
sterile distilled water was added and was placed
on the stirrer for 2 minutes to mix the spores
with water. The number of spores was measured
by a hemocytometer lam.
For anti-bacterial test, potato dextrose
(PDB) was prepared. Then these media
inoculated with bacterial and fungal
suspensions. Samples were incubated at
temperature on a shaker with 120 rpm.
were performed in a three iteration.
broth
were
spore
room
Tests
RESULTS AND DISCUSSION
Bacteria were cultured in NAS (Nutrient Agar
Sucrose) Media and after a week bacterial
suspension with a certain concentration was
prepared. Bacterial colony was added to the
1.5ml vials containing sterile distilled water then
the
concentration
was
adjusted
with
spectrophotometer.
Among fungi studied, some samples showed
significant results (Ekesi, 2005) indicating
significant presence of metabolic in them which
needs more investigation. Most of bacteria
isolates used in this study are gram-negative
bacteria which are plant pathogens.
Table-1. Bacterial and fungal cultures in broth media
Fungi
A.awamori
A.niveus
A.ferreus
A.usteus
A.sclerotiorum
A.ostianus
A.niger
A.corneus
A.fumigatus
A.parasitica
A.flavus
A.foetidus
P.moldavicum
P.mali
P.nigricans
P.piscarium
P.jenseii
P.janthinellum
P.corylophilum
P.cremeo-griseum
P.canescens
P.pinophilum
P.crustoseum
P.griseofulvum
P.chrysogenum
P.cammuni
P.aurantiigriseum
P.citrinum
P.chrysogenum5031
P.chrysogenum5033
P.chrysogenum5037
P.chrysogenum5071
P.chrysogenum5074
P. viridiflava
----------------------------------------------------------+++
+++
+++
---
259 |© 2015 Global Science Publishing Group, USA
Bacteria
X. campestris R. iranicus
+++
--+++
----+++
+++
--------+++
--+++
----------------+++
--------+++
+++
--+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
--+++
+++
--+++
+++
+++
+++
----+++
+++
+++
+++
+++
+++
+++
+++
E. coli
-------------------------------------------------------------------
Biolife | 2015 | Vol 3 | Issue 1
Seyedeh. Fatemeh. Miri et al
As you may observe in table 1, most of types
were not capable of controlling bacteria P.
viridiflava, except isolates of (5071, 5037 and
5033) from type P. chrysogenum. Regarding
bacterium R. iranicus we can claim that only P.
piscarium, P. corylophilum, P. griseofulvum and
P. crysogenum were not able to control this
bacterium and other types produced metabolic
which were able to control this bacterial and did
not allow growing up. Bacteria X. camstris py.
Campestris which is a key plant bacterial disease
showed significant results. Following types were
able to control this key bacterium: P.
chrysogenum 5037, P. chrysogenum 5071, P.
chrysogenum 5074, A. foetidus, P. mali, P.
crustoseum, P. citrinum, P. chrysogenum 5031,
A. awamori, A. ferreus, A. ostianus and A. niger.
Regarding E. coli bacteria, none of fungi could
control this bacterium. Maybe we can argue that
the metabolic with ability of removing this
bacterium was not produced which paved the
way for easy growth of this bacterium. Based on
the results mentioned we can argue that these
fungi do have the ability of controlling some of
the bacteria in soil. Since these bacteria produce
diseases in plants, presence of these fungi in soil
is important for reducing losses. Natural
compounds with pharmaceutical uses, played an
important role to treat human diseases and
microbial environments are important sources of
natural active agents (Newman D.J. et al. 2003).
Many compounds that are commonly used in
wide ranges are obtained by microbial
fermentations or chemical modifications of
biological compounds (Pranitha et al, 2014 and
Donadio S. et al. 2002). In this context,
fermentation is an efficient process for
secondary metabolites production which could
not be extracted from plants and animals and
even could not be produced by genetic
manipulations (Demain A.L. 2000). Two
antibiotics with new antifungal properties were
isolated from Aspergillus fumigates isolates
(Mukhopadhyay T. et al. 1987). In an effort to
find cell cycle inhibitors with microbial origins,
diketopiperazin was isolated from culture
fermentation of A. fumigates fungus (Cui C. B.
et al. 1997). Mentioned substances may play a
role in bacterial control of Penicillium species
260 |© 2015 Global Science Publishing Group, USA
Copyright@2014
tested in our experiments, which requires further
investigations about these issues in the future.
Figure-1. Positive and negative results of
broth media cultivation test, from right to
left.
CONCLUSION
Part of the events that occur in the soil could be
simulated at laboratory conditions. In this study,
results indicated several species of Penicillium
have the ability to control some plant pathogenic
bacteria. Subsequent studies regarding beneficial
and harmful metabolites of microorganisms
could be promising.
ACKNOWLEDGEMENTS
Authors are thankful to Mojtaba Najafi for
encouraging to the research during the
completion of the undertaken work.
REFERENCES
1. Alexopoulos CJ. & Mims CW. M. B. 1996.
Introductory Mycology. John Willey and
Sons. Inc. New York 869.
2. Ando K., Suzuki S., Seaki T., Tamura G.
& Arima K. 1969. Funiculosin, a new
antibiotic.Isolation, biological and chemical
properties. J. Antibiot. 22: 189–194.
3. Blondelle S. E., & Houghten R. A. 1996.
Trends in biotechnology. 14: 60-65.
4. Brands M., Endermann R., Gahlmann R.,
Krueger J., Raddatz S., & Stoltefu J.
2002. Novel antibiotics for the treatment of
Biolife | 2015 | Vol 3 | Issue 1
Seyedeh. Fatemeh. Miri et al
Grampositive bacterial infections. J Med
Chem. 45: 4246-53.
5. Butler MS., & Buss AD. 2006. Natural
products - the future scaffolds for novel
antibiotics Biochem Pharmacol. 71: 919-29.
6. Cui C. B., Kakeya H., & Osada H. 1997.
Novel mammalian cell cycle inhibitors,
cyclotryprostatins A-D, produced by
Aspergillus fumigatus, which inhibit
mammalian cell cycle at G2/M phase.
Tetrahedrom. 53: 59–72.
7. Cutler H. G., Arrendale R. F., Cole P.D.,
& Cox R. H. 1989. 3, 7-Dimethyl-8hydroxy-6-metoxy-isochroman
from
Penicillium corylophilum. Plant growth
regulatory activity. Agric. Biol. Chem. 53:
1975–1977.
8. Coates ARM., & Hu Y. 2007. Novel
approaches to developing new antibiotics for
bacterial infections. Brit J Pharmacol. 152:
1147-54.
9. Demain AL., & Elander RP. 1999. The blactam antibiotics: past, present, and future.
Antonie van Leeuwenhoek 75: 5-19.
10. Demain A. L. 2000. Small bugs, big
business: the economic power of the
microbe. Biotechnol.Adv. 18: 499-514.
11. Donadio S., Monciardini P., Alduina R.,
Mazza P., Chiocchini C., Cavaleti L., Sosio
M., & Puglia A.M. 2002. Microbial
technologies for the discovery of novel
bioactive metabolites. J. Biotechnol. 99:
187–198.
12. Ekesi N. K., Maniania Mohamed S. A., &
Lux S. A. 2005. Biol. Control. 35: 83-91.
13. Gandhimathi R., Arunkumar M., Selvin
J., Thangavelu T., Sivaramakrishnan S.,
Kiran GS. 2008. Antimicrobial potential of
sponge associated marine actinomycetes. J
Mycol Med. 18:16-22.
14. Grabley S., Granzer E., Hutter K.,
Ludwig D., Mayer M., Thiericke R., Till
G., Wink J., Philipps S., Zeeck A. 1992.
Secondary
metabolites
by
chemical
screening. Decarestrictines, a new family of
inhibitors of cholesterol biosynthesis from
Penicillium Strain description, fermentation,
isolation and properties. J. Antibiot. 45: 5665.
261 |© 2015 Global Science Publishing Group, USA
Copyright@2014
15. Gunatilaka A. A. L. 2006. Natural products
from
plant-associated
microorganisms:
distribution, structural diversity, bioactivity,
and implications of their occurrence. J Nat
Prod. 69: 509-26.
16. Jeong M. S., Park J. S., Song S. H., &
Jang S. B. 2007. Characterization of
antibacterial nanoparticles from the Scallop.
P. yessoensis. Biosci Biotecnol Biochem. 71:
2242-2247.
17. Kossem A., & Nannipieri P. 1995. Soil
cellulase activity. In Methods in Applied Soil
Microbiology and Biochemistry.
18. Kurobane I., Hutchinson R., & Vining L.
1981. The biosynthesis of fulvic acid, a
fungal metabolite of heptaketide origin.
Tetrahedron Lett. 22: 493-496.
19. Lockwood N. A., & Mayo K.H. 2003. The
future for antibiotics. Bacterial membrane
disintegrators. Drugs of the Future. 28: 91123.
20. Lotfi A., TajickGhanbary M. A, Ranjbar
G. A., & Asgharzadeh A. 2010. African
Journal of Biotechnology. 9: 4211-4216.
21. Luzhetskyy A., Pelzer S., & Bechthold A.
2007. The future of natural products as a
source of new antibiotics. Curr Opin Invest
Drugs. 8: 608-13.
22. Maestro B., and Sanz J. M. 2007. Novel
approaches
to
fight
Streptococcus
pneumoniae. Recent patents on anti-infective
drug discovery. 2: 188-96.
23. Maskey R. P., Grun-Wollny I., & Laatsch
H. 2003. Isolation, structure elucidation and
biological activity of 8-O-methylaverufin
and 1, 8-Odimethylaverantin as new
antifungal
agents
from
Penicillium
chrysogenum. J. Antibiot. 56: 488-491.
24. McLaughlin D. J., McLaughlin E. J., & P
(eds) L. 2001. The Mycota. Volume VIII:
part B. Systematics and Evolution,
(Springer-Verlag,Berlin).
25. Mukhopadhyay T., Roy K., Coutinho L.,
Rupp R. H., & Ganguli B. N. 1987.
Fumifungin, a new antifungal antibiotic from
Aspergillus fumigates Fresenius 1863. J.
Antibiot. 40: 1050-1052.
26. Nam Y. J., Kim K. H., Know Y. J., Han Y.
M., Son H. K., Lee C. U., Choi D. J.,
Know M. B. 2000. 8-O Methyl
Biolife | 2015 | Vol 3 | Issue 1
Seyedeh. Fatemeh. Miri et al
sclererotiorinamine, Antagonist of the Grb2SH2 Domain, Isolation from Penicillium
multicolor. J. Nat. Prod. 63: 1303–1305.
27. Newman D. J., & Cragg G. M. 2007.
Natural products as sources of new drugs
over the last 25 years. J Nat Prod. 70: 46177.
28. Newman D. J., Cragg G. M., Snader K. M
.2003. Natural products as source of new
drugs over the period 1981–2002. J. Nat.
Prod. 66: 1022–1037.
29. Nielsen J., & Smedsgaard J. 2003. Fungal
metabolite screening: database of 474
mycotoxins and fungal metabolites for
dereplication
by
standardized
liquid
chromatography–UV–mass
spectrometry
methodology. J. Chromatogr. A 1002: 111–
136.
30. Pranitha, V, Krishna G and M.A Singara
Charya. 2014. Evaluation of antibacterial
and anifungal activity of fruiting body
extracts of Trametes versicolar. 2(4):11811184.
Copyright@2014
31. Rousk J., & Baath E. 2007. Fungal biomass
production and turnover in soil estimated
using the acetate-in-ergosterol technique.
Soil BiolBiochem. 39: 2173-7.
32. Sharif M. 2009. American j of Agric and
Biol Sciences. 4: 152-155.
33. Singh P.D., Johnson J.H., Aklonis C.A.,
O’Sullivan J. 1986. SQ 30,957, a new
antibiotic
produced
by
Penicillium
funiculosum. J. Antibiot. 39: 1054-1058.
34. Singh S. B., & Barret J.F. 2006. Empirical
antibacterial drug discovery Foundation in
natural products. BiochemPharmacol. 71:
1006-15.
35. Sofia M. J., & Boldi A. M. 2006. In search
of novel antibiotics using a natural product
template approach Combinatorial Synthesis
of Natural Product-Based Libraries. 185-207
36. Strobel G., Daisy B., Castillo U., & Harper
J. 2004. Natural products from endophytic
microorganisms. J Nat Prod. 67: 257- 68.
****
262 |© 2015 Global Science Publishing Group, USA
Biolife | 2015 | Vol 3 | Issue 1